Basal keratinocytes are undifferentiating, proliferation-competent cells that terminally differentiate to produce the suprabasal epidermal layers. Differentiation can be induced by a variety of agents and involves coordinated activation of a number of genes. Extracellular calcium level is an important regulator of keratinocyte differentiation. Increased extracellular calcium promotes increased differentiation. Recent studies suggest that the extracellular calcium-sensing receptor (CaR), a G protein-coupled receptor, is involved in sensing changes in extracellular calcium and mediating the signal for differentiation. CaR mRNA and protein are expressed in the epidermal suprabasal layers - especially in the granular layer. However, the mechanism by which the CaR induces growth arrest and differentiation is not defined. We have shown that the CaR inhibits cell growth in renal epithelial cells. Thus, we are well positioned to study this protein in keratinocytes. The studies outlined in this pilot and feasibility proposal are designed to define the signaling pathways by which the CaR promotes keratinocyte growth arrest and differentiation. Specific Aim 1 is designed to define the effects of CaR on keratinocyte growth. These studies will test the hypothesis that the CaR acts by a specific, receptor-dependent mechanism to inhibit keratinocyte growth and induce differentiation. We will use non-functional, consitutively-active, and wild-type forms of the CaR to examine the effects of receptor inactivation and activation upon keratinocyte proliferation, and differentiation. We will also use receptor-specific pharmacologic agents to manipulate CaR activity. We will monitor effects on cell proliferation and expression of genes that are normally activated during differentiation, etc. Specific Aim 2 is designed to determine which signaling pathways mediate these responses. We will test the hypothesis that specific MAPK signaling pathways mediate the differentiation promoting effects of the CaR. We will also test the roles for phosphatases and filamin, a cytoskeletal scaffolding protein that interacts with the CaR. Thus, we will monitor the effects of constitutively-active, dominant-negative and wild-type CaR expression on the level and activity of the mitogen-activated protein kinases. Understanding the mechanism by which the CaR induces keratinocyte growth arrest and differentiation will provide fundamental new insights regarding the function of this receptor in epidermis, and may provide a rationale for treating patients with hyperproliferative skin diseases with agents that modulate CaR activity.